George Theodoris (ez003450 at bullwinkle.ucdavis.edu) wrote:
: Has anyone else had a problem with degradation of plasmid preps?
: When I first started in my current lab I was having a terrible problem
: with having my plasmids preps degrade in an unusual way. When the plasmid
: degraded it ran at the same length as RNA runs so at first I thought the
: RNAse I was using was not working.
: I found that several steps eliminated the problem:
: 1) Growing my Bacteria in LB rather than TB
: 2) Reducing the growing time to no longer than 14 hours
: 3) Switching from a boiling prep to alkali-lysis
: 4) Resuspending the DNA at a concentration not greater than
: 1microgram/1microliter
: Now someone else in my lab is having the same problem and I don't know
: what to tell her since she basicly follows the above steps except that
: she resuspends her DNA at a higher concentration. My experience was that
: the more concentrated the DNA was the more it was degraded. Extracting
: with Phenol-Chloroform did not help, but purifying the DNA on CsCl/EtBr
: usually did the trick. Has anyone else experienced this strange problem?
I had a similar problem a while back and found that the problem was with
the genotype of the bacteria. If the bacteria is HB101 it has the
wild-type endA gene. I switched to JM109 and the problem went away.
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